An apparatus for forming an array of deposits on a substrate is disclosed. The apparatus may include a stencil capable of releasable attached to the substrate and having an array of openings and at least one alignment mark. The apparatus may further include a high throughput deposition printer aligned with the stencil to form an array of deposits on the substrate. The array of deposits may be aligned with the array of openings through the at least one alignment mark and an optional alignment device. Methods of manufacturing the stencil and using it to generate multiplexed or combinatorial arrays are also disclosed.
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10. A method for printing an array of biomolecular deposits with nanoscale resolution on a substrate, the method comprising:
placing a parylene stencil over the substrate, the parylene stencil including an array of openings having at least one dimension of no more than 200 nanometers and at least one alignment mark;
aligning a deposition printer to the parylene stencil through the at least one alignment mark;
printing a first array of biomolecular deposits under hydrated conditions on the substrate through the array of openings in the parylene stencil by the deposition printer, the array of biomolecular deposits on the substrate having nanoscale resolution; and
removing the parylene stencil from the substrate.
1. An apparatus for printing an array of biomolecular deposits with nanoscale resolution on a substrate, comprising:
a parylene stencil including an array of openings and at least one alignment mark, the parylene stencil being capable of being releasably attached to the substrate, each of the openings in the array having at least one dimension of no more than 200 nanometers; and
a deposition printer aligned with the parylene stencil to print an array of biomolecular deposits under hydrated conditions on the substrate through the openings of the parylene stencil, the array of biomolecular deposits on the substrate having nanoscale resolution, the array of deposits from the printer being aligned with the array of openings in the stencil through the at least one alignment mark.
16. A method for printing a combinatorial array of deposits on a substrate with nanoscale resolution, the method comprising:
placing a parylene stencil over the substrate, the parylene stencil including an array of openings each having at least one dimension of no more than 200 nanometers;
printing a first array of deposits on the substrate through the array of openings in the parylene stencil, the first array of deposits on the substrate having nanoscale resolution;
printing a second array of deposits on the first array of deposits through the array of openings in the parylene stencil, at least one of the first and second arrays of deposits being multiplexed;
generating the combinatorial array of deposits on the substrate by allowing the first array of deposits to interact with the second array of deposits; and
removing the parylene stencil from the substrate.
2. The apparatus of
3. The apparatus of
4. The apparatus of
5. The apparatus of
6. The apparatus of
8. The apparatus of
11. The method of
providing an etching mask including a first mask and a second mask over a parylene layer such that the second mask is positioned between the parylene layer and the first mask;
forming an array of openings in the etching mask by a first etching process; and
transferring the array of openings from the etching mask to the parylene layer by a second etching process to provide the parylene stencil, the second mask being more resistant to the second etching process than the first mask.
13. The method of
15. The method of
17. The method of
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This application is a U.S. National Stage filing under 35 USC §371 of International Patent Application No. PCT/US09/44493 filed on Aug. 5, 2010, which claims priority under 35 USC §119(e) to U.S. Provisional Application Ser. No. 61/247,254 filed on Sep. 30, 2009, and U.S. Provisional Application Ser. No. 61/213,570, filed on Aug. 5, 2009.
The U.S. Government has an active license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of Grant No. R01DC007489-01A1, awarded by the National Institutes of Health.
1. Technical Field
Apparatus and methods for high-throughput formation of an array of deposits on a substrate with improved resolution and pattern fidelity are disclosed. In particular, this disclosure relates to the manufacture and use of a removable stencil aligned with a high-throughput deposition printer to control the location and dimension of a single, multiplexed, or combinatorial array of deposits on the substrate.
2. Description of the Related Art
Methods and apparatus for depositing arrays of substances with smaller dimensions (i.e. micro- or even nano-scales) on a substrate have been the central focus of a number of technological fields including electronics, optics, chemistry and biochemistry. In particular, biomolecular arrays with nano-scale features are useful for applications such as tissue engineering, cell cultures, and studying subcellular receptor-ligand interactions and intracellular processes. For example, cell behavior such as differentiation, adhesion and proliferation, can be regulated by patterned arrays of extracellular matrix (ECM) proteins with different feature shapes, sizes and spacings. Reducing the feature size of patterned ECM proteins to sub-200 nm dimension can help to elucidate the role of ECM proteins in forming focal adhesions with single-molecule resolution.
Furthermore, there is a growing interest to integrate biological and chemical functionalities with miniaturized sensor devices whereby accurate spatial positioning and alignment are crucial, such as nano-wire sensors, chemical field-effect transistors, nano-electromechanical sensors, and diffraction based antibody gratings. The diversity of protein molecules and their combinations present in nature requires the highly multiplexed capability of arrays to study the plethora of possible antagonistic and synergistic interactions between receptors and ligands. Hence, it is typical for hundreds or thousands of different biomaterial samples and their replicates to be patterned on a large area array and afterwards, allow for biomolecular ligands or cells to interact with the patterned surface.
Recent advances in printing and lithography have enabled the generation of patterned arrays with smaller features, some even with nano-scale resolutions. Exemplary array patterning techniques may include micro-contact printing (μCP) and atomic-force microscopy (AFM) based methods such as dip-pen lithography (DPN). While DPN is able to achieve nano-scale resolution down to tens of nm, the method is not easily scalable to print thousands of different biomolecules in a high-throughput fashion. Moreover, the final shape and size of deposits printed using DPN are controlled by surface hydrophobicity and chemistry. DPN may require extended length of time required to pattern large areas >1 cm2 even if multiple pens are used. μCP, on the other hand, may generate replicate arrays more rapidly but the elastomeric polymer stamps can deform with pressure and swell in aqueous conditions, resulting in relatively non-uniform nano-scale features. Additionally, μCP may not be suitable for generating arrays of superimposed depositions of multiple different types of samples as repeated alignment of elastomeric stamps with nano-scale precision may be difficult to achieve. Finally, the dimension, resolution, shape and reproducibility of deposits printed using current conventional printing methods depend on many factors such as surface chemistry, hydrophobicity and printing buffer and thus there is a need for robust and efficient printing apparatus and methods with better resolution and pattern fidelity.
Use of a polymer stencil in the deposition of micro-scale arrays is also known in the art. Specifically, a pre-defined array of micro-scale openings may be created photolithographically in a photoresist mask, and may be transferred into the polymer stencil through an etching process, which in turn is placed on the substrate for deposition of biomolecular samples through the micro-scale openings. Removal of the polymer stencil leaves the array of samples with defined micro-scale features on the substrate. Exemplary polymeric materials for making the stencil includes parylene, which is biocompatible and has been used as a template for patterning biomolecular arrays with features >1 μm, such as creating large area arrays of single-cells, proteins, nucleic acids, and lipid bilayers.
However, existing polymer stencil technologies typically rely on bath application of one type of sample onto the whole surface. Moreover, existing polymer stencil technologies may also be limited by the dimensions of the openings created in the stencil. Finally, high-throughput deposition of multiplexed combinatorial samples using polymer stencil technologies has yet to be developed.
This disclosure generally relates to methods and apparatus for high-throughput deposition of an array of one or more substances on a substrate with improved resolution and pattern fidelity, and particularly relates to the make and use of a removable stencil with an array of predefined openings to control the location and dimension of the substances deposited on the substrate. The disclosed apparatus and methods may reproducibly refine the imperfect deposits originally generated by the printer into well-defined deposits with improved resolution and pattern fidelity through the use of the removable stencil.
According to one aspect of this disclosure, an apparatus for forming an array of deposits on a substrate is disclosed. The apparatus includes a stencil releasably attached to the substrate and having an array of openings and at least one alignment mark. The apparatus further includes a high throughput deposition printer aligned (e.g. through an optional alignment device) with the stencil to form an array of deposits on the substrate. The array of deposits may be aligned with the array of openings through the at least one alignment mark.
According to another aspect of this disclosure, a method for generating an array of deposits on a substrate is disclosed. The method includes the steps of placing over the substrate a polymer stencil having an array of openings and at least one alignment mark; aligning a high throughput deposition printer to the polymer stencil through the at least one alignment mark; generating a first array of deposits on the substrate through the array of openings by the printer; and removing the polymer stencil from the substrate.
According to another aspect of this disclosure, a method for generating a combinatorial array of deposits on a substrate is disclosed. The method includes the steps of placing over the substrate a polymer stencil having an array of openings; generating a first array of deposits on the substrate through the array of openings; generating a second array of deposits on the substrate through the array of openings; and removing the polymer stencil from the substrate. At least one of the first and second arrays is multiplexed and the first and second arrays of deposits may be capable of interacting with each other. As used in this disclosure, the term “multiplexed array” refers to a layer of array of deposits in which at least two deposits are different from each other in terms of their chemical and/or biological properties. Accordingly, the term “uniplexed” refers to a layer of array of deposits in which all deposits are identical in terms of their chemical and/or biological properties.
According to another aspect of this disclosure, a method for forming a polymer stencil is disclosed. The method may include the steps of providing an etching mask over a polymer layer, forming at least one opening in the etching mask by a first etching process, and forming at least one opening in the polymer layer by a second etching process. The etching mask may include a first mask and a second mask positioned between the polymer layer and the first mask. The second mask may be more resistant to the second etching process that the first mask.
Other advantages and features of the disclosed apparatus and methods will be described in greater detail below. It will also be noted here and elsewhere that the apparatus or method disclosed herein may be suitably modified to be used in a wide variety of applications by one of ordinary skill in the art without undue experimentation.
For a more complete understanding of the disclosed method and apparatus, reference should be made to the embodiments illustrated in greater detail in the accompanying drawings, wherein:
It should be understood that the drawings are not necessarily to scale and that the disclosed embodiments are sometimes illustrated diagrammatically and in partial views. In certain instances, details which are not necessary for an understanding of the disclosed apparatus or method which render other details difficult to perceive may have been omitted. It should be understood, of course, that this disclosure is not limited to the particular embodiments illustrated herein.
Referring now to
The positioning device 16 may include a position rail 17 extending in an X-direction and movable in a Y-direction parallel to the support plate 14. The positioning device 16 may also include a position arm 18 having a first end 19 attached to, and movable in the X-direction along, the position rail 17 and a second end 20 upon which the print head 15 is mounted. The position rail 17 and position arm 18 may be operatively connected to one or more motors (not shown) and controlled by a processor (not shown) so that the position of the print head 15 relative to the substrate 11 on the support plate 14 can be precisely controlled.
One non-limiting example of the printer 13 suitable for use in this disclosure is a high-throughput Marathon Inkjet Microarrayer printer (provided by ArrayJet, Midlothian Innovation Centre, Pentlandfield, Roslin, EH25 9RE, Scotland—UK), which is capable of generating deposits directly on the substrate with micrometers resolution. However, the sizes and shapes of the deposits can vary from spot to spot, depending on factors such as printing buffer conditions and surface chemistry/hydrophobicity. Deposits with smaller feature sizes, such as nanometer resolutions, may be difficult to achieve by direct inkjet printing.
To improve the reproducibility and pattern fidelity of the deposits generated by the high-throughput printer 13, and/or to achieve deposits with nano-scale features, the polymer stencil 12 is provided on the top surface of the substrate 11 during printing and removed from the substrate once the deposits are formed. The average diameter of the original printed deposit may be larger than the average diameter of the opening or the array of openings in the polymer stencil, such that the printed deposit completely covers the opening or the array of openings. A non-limiting example of the polymer stencil 12 is illustrated in
As illustrated in
Turning back to
One feature of the parylene stencils made by the disclosed method is that they are substantially pinhole-free and relatively inert to chemical reactions. Another feature of the parylene stencils made by the disclosed method is that they exhibit limited or no swell in aqueous solutions, which may make them suitable for patterning relatively large area with uniform arrays of biomolecules with improved pattern fidelity. Moreover, it is contemplated that the parylene stencils made by the disclosed method may be used to pattern biomolecular arrays under hydrated conditions, which preserves the conformation and functionality of those biomolecules that are sensitive to moisture. It is to be understood that other polymer materials, natural or synthetic, may also be used instead of, or in addition to, the parylene material disclosed above.
The etching mask 32 may include a first mask 33 and a second mask 34 positioned between the polymer layer 31 and the first mask 33. In one embodiment, the first mask 33 may include an electron beam resist material (e.g. ZEP520 resist by Zeon Chemicals, 4111 Bells Lane, Louisville, Ky.). The second mask 34 may include a metal material, such as aluminum. The second mask 34 may be deposited on the polymer layer 31 by thermal or electron beam evaporation, and the first mask 33 may be formed on the second mask 34 by spin-coating. It is to be understood that the coating and deposition methods disclosed above are exemplary and should not be interpreted as limiting the scope of this disclosure.
In order to form nano-scale openings on the polymer layer 31, the openings 23 are first formed in the etching mask 32 through a first etching process, as illustrated in
In step III, the array of openings 23 formed in the first mask 33 may be further transferred to the second mask 34. When the second mask 34 is made of a metal, the array of openings 23 may be created in the second mask 34 through reactive-ion etching. Thus, in some embodiment, the first etching process may include EBL in the first mask 33 and reactive-ion etching in the second mask 34 (steps II and III). Further, when the second mask 34 is made of a metal, a metal oxide may be formed on the surface of the second mask 34 and affect the reactive-ion etching of the second mask 34, in which case the first etching process may further include an additional step of applying high voltage (e.g. 450V) on the second mask 34 to remove the metal oxide thereon. As indicated in
In step IV, the array of openings 23 formed on the second mask 34 is transferred to the polymer layer 31 through a second etching process. When the polymer layer 31 is made of parylene-C, the second etching process may also include reactive-ion etching, albeit at a less harsh condition than the reactive-ion etching of the second mask 34, to preserve the structural integrity of the well-defined array of openings on the second mask 34. After the array of the openings 23 are transferred to the polymer layer 31 (now the polymer stencil 12), the second mask 35 may be removed from the polymer stencil 12 in step V, leaving only the polymer stencil 12 on the substrate 11. Because the second mask 34 is more resistant to the second etching process than the first masks 33, the array of openings 23 formed with high-resolution (e.g. with nano-scale features) may be transferred to the polymer stencil 12 with high fidelity, as described with greater details in the examples below.
In a non-limiting example of the method disclosed above, a thin layer of parylene-C (obtained from Uniglobe Kisco, 707 Westchester Avenue, Suite 207, White Plains, N.Y. 10604, USA) was conformally vapor coated onto a 4″ oxidized silicon wafer using a Parylene Labcoater (obtained from Specialty Coating Systems, 7645 Woodland Drive, Indianapolis, Ind. 46278, USA). Thereafter, an aluminum thin film (e.g. 15 nm) was thermally evaporated on top of the parylene. Zeon ZEP-520 resist was spun to a 100 nm thickness on top of the aluminum film, baked at 90° C., and exposed by electron beam lithography (EBL) using the JEOL JBX-9300FS to generate an array of openings with nano-scale features.
The resist was then developed in n-amyl acetate for 45 seconds and rinsed for 30 seconds in methyl isobutyl ketone and 2-isopropanol. The array of openings in the resist was transferred into the aluminum film using reactive ion etching with Cl2/BCl3/CF4 chemistry in a PlasmaTherm® 740 etcher. Finally, the nano-scale openings were transferred into the parylene using the patterned aluminum film as a hard etch mask and oxygen plasma reactive ion etching with the Oxford PlasmaLab® 80+ etcher. The aluminum film was dissolved in MIF300 developer (obtainable from e.g. MicroChem, 90 Oak St., Newton, Mass. 02464, USA), leaving the parylene stencil on the wafer. As illustrated in
Turning now to
Thus, the disclosed method is capable of generating an array of openings with nano-scale resolution (e.g. no more than about 200 nm) in a high-throughput nanofabrication process, an achievement heretofore unknown. In particular, the use of the second mask may promote the transferring of openings with nano-scale features from the first mask 33 to the polymer stencil 12 with high fidelity that is not available to existing single-mask processes. Without wishing to be bound by any particular theory, it is contemplated that such high fidelity may be at least partially attributed to the second mask being more resistant to the etching condition of the polymer layer. In the above-described process, for example, the aluminum mask may form a stable oxide layer in the presence of oxygen. This aluminum oxide layer maintains etch anisotropy, which prevents lateral widening of the features during pattern transfer into the parylene layer. Although such lateral widening may be insignificant during existing processes of generating micrometer-sized openings in parylene with a photoresist or electron beam resist etch mask, it may prevent accurate transferring of nanometer-sized openings from the resist material to the parylene layer. Moreover, in some cases, the oxide layer may also prevent the aluminum layer from being etched efficiently by the chlorine etch chemistry during the pattern transfer from the resist layer into the aluminum mask. To remove the aluminum oxide layer, a high power of 450V may be applied to the aluminum mask before the openings are transferred into the aluminum mask. This step may also facilitate the removal of the resist layer. Again, without wishing to be bound by any particular theory, it is contemplated that the resolution and size of the nano-scale openings in the parylene stencil generated by the disclosed method may be a function of the etch selectivity, anisotropy, and/or aspect ratio of the etching steps in the nanofabrication.
Turning back to
As a non-limiting example, a patterned nano-array of fibronectin was deposited on a substrate using the parylene stencil 12 with high fidelity. Specifically, human fibronectin (obtained from Sigma-Aldrich) was dissolved in deionized water and diluted in phosphate buffered saline to 10 μg/mL. The diluted fibronectin was manually spotted onto the parylene template, incubated for 2 hours, and rinsed with water to remove excess unbound fibronectin. The parylene stencil was peeled off with tweezers under water to define an array of nanoscale fibronectin features. The array was kept in hydrated conditions throughout the patterning to preserve the 3D conformation and functionality of the protein.
As illustrated in
As schematically illustrated in
As illustrated in
In a further refinement, two or more layers of array of deposits that are capable of interacting with each other may be superimposed to generate combinatorial arrays of increasing complexity when at least one of the layers is multiplexed, another feature heretofore unknown. The interaction may include, but is not limited to chemical reactions, receptor-ligand bindings, enzyme-substrate interactions, interactions that generate fluorescence, chemiluminescence or color change, and combinations thereof.
As schematically illustrated in
Although purified proteins and antibodies were used in the examples disclosed herein, any desired substances, such as nucleic acids, cells and cell lysates, can be printed in combination or by itself. Arraying different combinations of biomolecular components using the disclosed process may be used in combinatorial screening of pharmaceutical compounds and responses of stem cells. The ability of the disclosed process to create combinatorial biomolecular nanoarrays may also facilitate the combinatorial study of synergistic and antagonistic properties of biomolecules in receptor-ligand interactions. Although two multiplexed arrays are used in the above-described example, the combinatorial array may also include one multiplexed array superimposed with a uniplexed array (e.g. bath-applied) or include more than two arrays superimposed with one another so long as at least one of the arrays are multiplexed.
It is to be understood, of course, that the disclosed methods and apparatus can be used to print a wide variety of materials, including the protein samples used in the above-discussed examples. Other biological or biomolecular substances suitable for use in the disclose methods and apparatus include, but certainly not limited to amino acids, peptides, oligopeptides, polypeptides, nucleotides, oligonucleotides, polynucleotides, saccharides, oligosaccharides, polysaccharides, lipids, carbohydrates, enzymes, steroids, metabolites, and derivatives thereof. Non-biological samples including inorganic, organic, metallic, and organometallic compounds may also be printed using the disclosed methods and apparatus.
While only certain embodiments have been set forth, alternative embodiments and various modifications will be apparent from the above descriptions to those skilled in the art. These and other alternatives are considered equivalents and within the spirit and scope of this disclosure.
Lin, David M., Craighead, Harold G., Tan, Christine P.
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